Depositing Liquids on Reagent-Containing Substrates
Many instruments have been developed to measure the quantity of analytes in biological samples, for example urine, blood, salvia, or extracts of mucus or tissue. Typically, a sample liquid is applied to a surface containing reagents that react with the analyte. The reagents produce a detectable response that is measured and related to the amount of the analyte. The surface usually will be either hydrophilic or hydrophobic in nature, e.g. filter paper compared to polystyrene. Some devices use combinations of surfaces, such as urinalysis strip tests that use hydrophilic filter paper pads on top of a hydrophobic polystyrene handle. In the typical test, a strip containing unreacted reagents is dipped, i.e. fully immersed in a liquid sample, and the reaction between the analyte in the sample and the reagents is measured, usually by optical methods. The unreacted reagents themselves may be water soluble or insoluble. They are deposited or immobilized and dried in a porous substrate. The substrate is attached or placed onto the supporting surface. Additionally, a liquid with or without reagents can be used during an assay. The liquid reagents can be applied to the surfaces of substrates already containing dried reagents, before, after or during the reaction with the analyte, typically being added after a sample has been applied. The volume of samples and reagents should be as small as possible for obvious reasons relating to cost and convenience. What is less obvious is that it is often difficult to obtain a uniform and accurate response when applying small amounts of liquid reagents or biological samples to surfaces containing reagents. The response of the analyte with reagents is smaller than the reaction area in smaller and less analyte is present.
The substrate can be used to amplify the reaction response. Thin films, e.g. membranes, can be immobilized with affinity reagents to allow capturing and concentration of reactants in read zones. Directing flow of liquids in a desired direction, e.g. laterally rather than vertically, can increase efficiency by increasing the number of fluidic exchanges between the liquid sample or reagent and the reaction zone. Each exchange allows further reaction of the analyte to occur, thereby amplifying the signal. Modification of the surface of the substrate allows reagents to be isolated in the reaction zone. Further, the nature of the surface itself can be used to increase the reactivity of the analyte, for example by increasing solubilization of reagents or to favor reactions with reagents on the surface.
Most biological samples and liquid reagents will have a significant water content and thus will be compatible with hydrophilic substrates and incompatible with hydrophobic surfaces. The sample and reagent liquids when dispensed spread rapidly across hydrophilic substrates and are repelled by hydrophobic substrates. The contact between the dispensed liquid and the reagents on the surface is made by direct dispensing onto the reacted or partially reacted area. However, when substrates are relatively hydrophobic, the dispensed liquid will form beads on the surface of the substrate that attempt to minimize their contact with the surface and therefore they do not spread uniformly over the reagent. Another difficulty associated with dispensing liquids is that the dried reagents may be either water soluble or water insoluble in nature. The insoluble dry reagents may not be readily accessible to the liquid samples, or soluble reagents may be dissolved and move with the liquid on the substrate. The reagents ideally should contact the sample uniformly, since the measurable response of the reagents to the sample, e.g. color development, should be uniform in order to obtain an accurate reading of the quantity of the analyte in the sample.
Another problem related to obtaining good contact between a dispensed liquid and a reagent on a surface is related to the physical nature of the samples. They vary in their physical properties such as surface tension, viscosity, total solids content, particle size and adhesion. Therefore, they are not easily deposited in consistent volumes uniformly over the reagent-covered substrate. Also, as the amount of the liquid sample is reduced, it becomes increasingly difficult to apply a consistent amount of a sample having varying properties to the reagents. In contrast, ink jet printing and the like rely on liquids developed for such uses and having consistent physical properties.
Deposition of droplets of liquid is a familiar operation. Examples include the ink jet-printer, either piezoelectric or bubble actuated, which forms print from the controlled deposition of multiple small droplets of about 2 to 300 μm diameter (typically 50 μm) containing from a few femtoliters to tens of nanoliters. Other methods of depositing small droplets have been proposed, which generally employ piezoelectric principles to create droplets, although they differ from typical ink jet printers. Examples are found in U.S. Pat. Nos. 5,063,396; 5,518,179; 6,394,363; and 6,656,432. Deposition of larger droplets (3-100 μl) through a syringe type pipette is known to be reproducible in diagnostic systems. This corresponds to single droplet diameters of about 2 to 6 mm. A commercial example of such pipette systems is the CLINITEK ALTAS® urinalysis analyzer. The droplet size can be greater or less than the nozzle size depending on the nozzle shape, pump type and pressures applied.
The problems discussed above are particularly observed when a liquid sample is dispensed as droplets onto a reagent-containing pad. It has been found that the interactions of the pad's surface and the reagents were creating inaccurate responses when the sample was added as a droplet, rather than completely covering the reagent pad by immersing the reagent pad (dipping it) into the sample liquid, as is frequently done. Large droplets on the order of 3 to 100 μL do not transfer into the reagent when the substrate is too hydrophobic and form a bubble on the surface. They overwhelm the reagent with excess fluid if the surface is hydrophilic. Smaller droplets, of a few femtoliters to tens of nanoliters, can also be a problem when deposited on a substrate that is too hydrophobic as they lack the volume to completely cover the surface area and will randomly aggregate in non-uniform patterns. Small drops also allow open spaces for migration of water-soluble reagents. These tiny droplets are also prone to evaporation of liquids and to formation of aerosols, which are considered to be biohazardous if comprised of urine or blood specimens. Thus, if a liquid is to be deposited as droplets on test pads, rather than dipping the pads in the sample, improvements were needed.
After contact between dispensed liquids and reagents is complete, the results may be read using one of several methods. Optical methods are commonly used, which rely on spectroscopic signals to produce responses. Results must be reproducible to be useful. Optical measurements are affected by the reagent area viewed and by the time allowed for the dispensed liquids and reagents to react. Formation of non-uniform areas within the field of view and changes in the amount of reaction time cause increased errors. For example, a measurement made of a sample or reagent which has spread non-uniformly across the substrate gives a different result each time it is read.
In co-pending U.S. patent application Ser. No. 11/135,928, published as U.S. 2006/0263902 A1, commonly assigned with this application, the inventors reported their methods of depositing biological fluids and reagents as fine droplets onto reagent-carrying substrates. They demonstrated that the reagent-carrying substrates behaved differently, depending on the water solubility of the reagents and the surface energy of the substrate, that is, whether the reagent-carrying substrates were hydrophilic or hydrophobic. Depositing large droplets, e.g. 1.7-20.4 μL, was shown to provide less accurate results than when small droplets of about 50 pL to 1 μL were deposited on reagent-carrying surfaces. The inventors also found that small droplets were absorbed by the hydrophobic substrates, while large droplets were not readily absorbed.
Water soluble reagents were shown to dissolve and move with a liquid as it spreads on a reagent-carrying surface. The inventors found that that non-uniform reagent response which such movement caused would be moderated by depositing small droplets.
Depositing of small droplets was done either by nozzles having many small openings or by single nozzles, which could be moved relative to the reagent-carrying substrate, or vice versa, to cover the desired area. The reaction of liquid samples with reagents on the substrate could be read as an average of the area covered by the sample or preferably by scanning the reaction area one spot at a time and averaging the results.
Deposition Liquids into Microfluidic Devices
Adding biological samples and associated liquids to microfluidic devices used for analysis of biological samples may be done with various techniques. Very small samples of blood, urine and the like are introduced into such devices, where they come into contact with reagents capable of indicating the presence and quantity of analytes found in the sample.
Problems associated with depositing biological samples and other liquids that may be needed for analyzing the samples have been discussed in U.S. patent application Ser. No. 10/608,671, published as U.S. 2004/0265172 A1. Particularly important requirements are the removal of air from the device as liquids are introduced and metering the amount of sample to be analyzed, and associated liquids, e.g. reagents, buffers, diluents and the like.
It has been found that, even after the problems just discussed have been overcome by proper design of the microfluidic device, measuring the amount of an analyte in a biological sample may not give the repeatability that one would like. In part, the problem relates to the variability inherent in these designs. First, the variability in the surface coating can cause liquids to creep over capillary stops or around reagent areas. This causes variations in the timing of liquid movements and the volumes reacted. Second, less experienced users can apply incorrect amounts of samples or reagents. Third, the internal dimensions of these microfluidic devices can differ from one chip to another when they are made in large quantities by low cost methods. The present inventors have found that such problems can be overcome, making significant improvements in the accuracy and repeatability of results.